LOCUS       sequence-430873-        6506 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The DuET construct encodes the named human GPCR engineered with a 
            cleaved hemagglutinin signal peptide, a 5' FLAG and a 3' 1D4 epitope
            tag optimized for expression in mammalian cells and proteomics 
            studies..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6506)
  AUTHORS   Dahl L, Kotliar IB, Bendes A, Dodig-Crnkovic T, Fromm S, Elofsson A,
            Uhlen M, Sakmar TP, Schwenk JM
  TITLE     Multiplexed selectivity screening of anti-GPCR antibodies.
  JOURNAL   Sci Adv. 2023 May 3;9(18):eadf9297. doi: 10.1126/sciadv.adf9297. 
            Epub 2023 May 3.
  PUBMED    37134173
REFERENCE   2  (bases 1 to 6506)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Adv."; 
            date: "2023-05-3"; volume: "9(18):eadf9297. doi"; pages: " 
            10.1126/sciadv.adf9297. Epub 2023 May 3"
COMMENT     Sequence Label: MRGPRX3-DuET
FEATURES             Location/Qualifiers
     source          1..6506
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     292..311
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(530..634)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(709..728)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        900..1279
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1280..1483
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1434..1454
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1528..1547
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1528..1546
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2762..2779)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2768..2992
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3038..3466
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3125..3144)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3335..3356
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3475..3495)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3480..3809
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3660..3795
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3722..3741
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3876..4670
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3930..3949)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4540..4559
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4844..4965
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4881..4900)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4935..4954
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5014..5030)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5014..5030)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5027..5049)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5038..5054
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5062..5068,5069..5086,5087..5092))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5107..5128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5245..5262)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5416..6004)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5496..5515)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6175..6506,1..460,461..529))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
       61 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
      121 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
      181 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
      241 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
      301 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
      361 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
      421 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
      481 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
      541 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
      601 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
      661 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
      721 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
      781 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacattt gcatgaagaa
      841 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
      901 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
      961 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     1021 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     1081 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     1141 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     1201 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     1261 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     1321 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     1381 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     1441 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga
     1501 acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg
     1561 ctagcgttta aacttaagct tagcgccacc atgaagacga tcatcgccct gagctacatc
     1621 ttctgcctgg tattcgccga ctacaaggac gatgatgacg ccagcatcga tatggacagc
     1681 actatacccg tgttgggtac cgaacttacc cctattaacg ggcgggagga aaccccttgt
     1741 tacaagcaga ccctttcatt taccggcttg acttgcatcg tgtctcttgt ggccctgacg
     1801 ggaaacgcag ttgttctttg gctgcttggc tgtaggatgc gacggaatgc cgtgtccatc
     1861 tacattctca accttgttgc cgcagacttc ctctttctca gcggacatat tatctgttcc
     1921 cccttgaggc tgatcaatat tcgccatccg attagcaaga tattgagtcc tgtaatgaca
     1981 ttcccttatt tcatcggctt gtctatgctg tcagccatct caactgaaag gtgcctcagc
     2041 atcctgtggc cgatttggta tcactgccgc agacccagat acctgtccag cgtaatgtgt
     2101 gtactcctgt gggcgctctc tctgctcaga agtatcctgg aatggatgtt ttgtgacttc
     2161 cttttttctg gagcgaatag cgtgtggtgt gagacctccg acttcataac aatcgcatgg
     2221 cttgtatttc tgtgtgtggt tctgtgtggc tcatctctgg ttctgctggt caggatcctc
     2281 tgcgggagtc gaaaaatgcc actgactaga ctctatgtta caatcctcct gacggtgctt
     2341 gtctttctcc tctgcggact gccctttgga atacagtggg cacttttttc caggattcat
     2401 ctggattgga aagtactttt ctgccacgtg cacctggtga gtatcttttt gagcgctctg
     2461 aactctagcg caaatccgat tatatatttt tttgtgggtt ctttccggca gcgccagaac
     2521 agacagaatc tcaaattggt tcttcagcgg gccctgcagg acacaccaga ggtggatgaa
     2581 ggaggcggat ggcttcccca ggagacactg gagctgtctg gcagcaggtt ggagcagatc
     2641 gataccggtg gacgcaccgg tgaattcgat gaggccagca ccaccgtgtc taagaccgag
     2701 acttctcagg tggcccctgc ctaactcgag tctagagggc ccgtttaaac ccgctgatca
     2761 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     2821 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     2881 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     2941 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     3001 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
     3061 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     3121 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     3181 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     3241 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     3301 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     3361 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     3421 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
     3481 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3541 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3601 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     3661 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     3721 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
     3781 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
     3841 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     3901 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     3961 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     4021 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
     4081 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     4141 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     4201 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     4261 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     4321 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     4381 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg
     4441 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     4501 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     4561 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     4621 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     4681 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     4741 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     4801 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     4861 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4921 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     4981 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     5041 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     5101 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5161 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5221 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5281 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5341 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5401 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5461 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5521 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5581 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5641 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5701 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5761 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5821 cttgaagtgg tggcctaact acggctacac tagaagaacg gtatttggta tctgcgctct
     5881 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5941 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     6001 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6061 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     6121 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6181 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6241 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6301 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6361 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6421 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6481 tgccattgct acaggcatcg tggtgt
//
